Production of molecular arrays

ABSTRACT

The subject invention pertains to a method for the production of an array of molecules immobilised on a substrate, comprising the deposition of the molecules from a micropipette containing them, onto the substrate, in a liquid environment, wherein the distance of the micropipette from the substrate is controlled in response to the ion current in the liquid. This method is particularly suitable for the deposition of biological molecules. The subject invention also pertains to an array of biological molecules deposited on a substrate.

FIELD OF THE INVENTION

[0001] Their invention relates to nanotechnology, and more particularlyto the production of molecular arrays.

BACKGROUND OF THE INVENTION

[0002] Currently there is a great interest in nanotechnology, themanipulation and study of matter at the nanometer scale (see a recentScience review, Nov. 24, 2000). There is a belief that, in the longterm, nanotechnology will lead to useful devices or structuresfunctioning on the nanometer scale. In recent years, the manipulation ofsmall molecules and atoms has been demonstrated; for example thescanning tunnelling microscope tip has been used to write “IBM” withxenon atoms and also to perform local catalysis on a metal surface. Theatomic force microscope (AFM) tip has also been used for so-called“dip-pen” nanolithography (Piner et al, Science 1999,28,3661), to writefeatures by contact with gold surfaces using thiol chemistry, down to10-20 nm feature size. However, these methods are not compatible withthe use of biological molecules and not straightforward to implement.This is because they require very clean surfaces and careful controlover the conditions of deposition.

[0003] To date, the only methods of note for manipulation of biologicalmolecules are optical tweezers, the atomic force microscope (AFM) andmicrofluidics. Optical tweezers have been applied to biologicalmolecules attached to micron-size beads. For example, tweezers have beenused to stretch DNA molecules. The AFM has been used to bring onemolecule on the tip into contact with a molecule on the surface. Boththese methods are limited to manipulating single molecules one at a time(for a review, see Nature Reviews 2000,1,130).

[0004] Microfluidics, using patterned polymer films, has been used toflow biological molecules down channels for separation and analysis. Ithas been used on large single DNA molecules flowing along a channel, formolecule by molecule analysis (Science 2000, 294,1536)). This method islimited in the range of possible applications and does not allow theopportunity to write in biological molecules.

[0005] Microcontact printing has also been used to create patterns ofbiological molecules on a surface by direct stamping. This is limited toone type of biological molecule and also has limitations in the featuresize possible.

[0006] DNA and peptide arrays, provided on a single chip, are importanttools in molecular biology. Their production, on a commercial scale,poses various problems.

[0007] The current methods for writing with biological molecules arebased on spotting using a pipette or piezoelectric delivery in air. Forexample, in the manufacture of DNA arrays, the feature size is about50-100 μm. The photolithographic method for DNA chip productiondeveloped by Affymetrix has a fundamental feature size limit, due todiffraction of about 250 nm. Moreover, this limit has not yet beenrealised due to inefficiencies in the chemistry.

[0008] Schreiber has recently described a simple protein array, obtainedby spotting proteins onto aldehyde-functionalised glass slides (Science2000, 289, 1760-1763). The spot resolution was 150 μm, and there weretechnical issues with keeping the protein solvated, and blocking offunreactive aldehydic sites. Thus, the manipulation and writing withbiological materials lag well behind that of atoms and small moleculesdue to the lack of suitable methods.

[0009] Scanning ion conductance microscopy (SICM) is a form of scanningprobe microscopy, to image the surface of living cells, e.g. at 50 nmresolution. The method is based on scanning a micropipette over thesurface of a cell and using the ion current that flows to an electrodeinside the pipette to maintain and regulate the distance from thesurface.

[0010] As disclosed in WO-A-00/63736, when the sample-pipette distanceis modulated, an additional modulated current is produced, which adds tothe DC current. The modulated signal only becomes significant when thepipette is close to the sample. This provides a robust, reliable methodof distance control and virtually eliminates problems in dc drift orchanges in ionic strength.

[0011] Zhang et al, J Vac. Sci. Technology 1999, B17(2), 269-272,discloses that a micropipette can be used to process microcircuits andmicrostructures on a substrate, by SICM. It is limited to conductingsurfaces and to electrochemical reactions. The micropipette tip-surfacegap is controlled by the ion current, without distance modulation. If asmaller voltage is applied, then less deposition takes place, but thisalso affects the position of the pipette with respect to the surface;since the current would be smaller, the micropipette would move awayfrom the surface to maintain constant current. Control is thus difficultif not impossible.

SUMMARY OF THE INVENTION

[0012] The present invention is based on the realisation that writingwith biological molecules can be achieved, by reducing the size of thepipette used in SICM, to the nanometer scale. Thus, a micropipette ofthe type used in SICM can apply biological molecules to a surface. SICMis well suited for this task since it has been developed for imagingcells in physiological buffer and hence is perfectly compatible withbiological materials. The pipette is a local and large reservoir of ahigh concentration of molecules allowing many applications. Themicropipette diameter is typically 50-1000 nm and is easily altered byadjustment of the pulling parameters used. Since the micropipette-sampledistance is controlled using the ion current, typically to themicropipette radius, this means that novel feature sizes of less than 1μm, e.g. 10-100 nm, may be achieved. The application is performedentirely in solution so that the characteristics of the flow out of thepipette and diffusion are the only factors affecting feature size (anymolecules not adsorbing locally onto the surface will get rapidlydiluted by diffusion into the bath). Furthermore, the method canstraightforwardly be extended to multiple applications by the use ofmulti-barrel micropipettes. Thus micropipette application of biologicalmolecules appears to have many potential advantages over other methods.

[0013] The novel method can be used to deposit biological molecules atspecific positions on a surface and also to control and monitor thedeposition, down to the level of single molecules. The self-assembly andspecific recognition possible with biological molecules offersintriguing possibilities for the production of novel nano structures andbiosensors. Furthermore, it may be possible to exploit the specificrecognition of biological molecules to directly print multiple copies ofa single master structure once it has been created.

[0014] The pulsed delivery of single-stranded DNA molecules through ananopipette has been demonstrated. The conical geometry of the pipetteleads to a localized electric field since all the potential drop occursin the tip region. Pulsatile delivery of DNA molecules can be achievedin an experimentally simple way with high precision by controlling theapplied voltage. Single-molecule detection and fluorescence correlationspectroscopy in the nanopipette enabled determination of the number ofmolecules delivered. Anomalous slow diffusion of the DNA molecules inthe pipette has also been observed. This nanopumping technique may havepotential applications in local drug delivery, and nanofabrication ofbiomolecules on surfaces in aqueous environments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1A is a schematic view of a device that can be used in theinvention.

[0016]FIG. 1B is schematic of an experimental setup. SPAD: single photoncounting avalanche photodiode, MCS: multichannel scalar.

[0017]FIG. 2 shows the results of pulsatile delivery of DNA molecules byvoltage modulation at 10 Hz. Highly repetitive pulses have beenobserved. Optimal pulse can be obtained by manipulating the modulationamplitude and duty cycle. The laser was focused about 0.5 mm in front ofthe tip opening. The MCS integration time bin width is 0.2 ms. (A)Modulating from −0.1V to 0.6V with positive voltage duration of 30 ms,about 150±20 molecules were detected per pulse; (B) Modulating from−0.1V to 1.5V with positive voltage duration 5 ms, about 460±50molecules were detected per pulse.

[0018]FIG. 3 shows single-molecule detection of a rhodaminegreen-labelled 20-mer ss-DNA exiting from a nanopipette. The laser focuswas centered at the opening of the pipette tip. The MCS bin width is 1ms. (A) At different applied voltages; (B) Voltage modulation from −0.1Vto 1.0V.

[0019]FIG. 4: A) Pulse patterns of fluorescence at different modulationamplitude. The modulation frequency is 10 Hz and the duty cycle is 0.3.The MCS bin width is 0.2 ms. Ten cycles were averaged to reduce the shotnoise. B) The number of DNA molecules detected per pulse as a functionof applied voltage V_(on). A linear relationship was observed.

[0020]FIG. 5: A) Schematic of the experiment to measure the flow of DNAmolecules out of the pipette. The pipette is filled with 100 nMrhodamine green labelled, single stranded (ss) DNA molecules. When apositive voltage is applied to the Ag/AgCl electrode in the bath, themolecules are driven out of the pipette. Fluorescence under 488 nm laserexcitation (100 μW) can be detected using a microscope and a photoncounting avalance photodiode. The blue area depicts the confocal laserillumination. B) Fluorescence intensity of rhodamine green labelledssDNA as a function of the electrode potential. The right y-axis showsthe flux of molecules leaving the tip, estimated by single moleculecounting experiments. C) Schematic of the deposition of ssDNA onto thestreptavidin coated glass surface in phosphate buffer solution (10 mMNa₂HPO₄, 137 mM NaCl). The DNA molecules are labelled with biotin andrhodamine green. The ion current flowing through the tip is used tocontrol the tip-surface distance and the DNA is immobilized by thestreptavidin-biotin binding. D) Detection of the written structures byscanning confocal fluorescence microscopy, exciting the rhodamine greenlabelled DNA at 488 nm and recording the fluorescence centred at 530 nm.

[0021]FIG. 6: A) Fluorescent image of dots written in DNA on thestreptavidin coated surface with increasing deposition time of 5, 10,20, 40, 80, and 160 s, the image size is 25 μm×25 μm, the colour mapused to display the intensity is given in FIG. 2B. B) Peak intensity(blue squares) and total intensity (black circles) of the dots in FIG.2A as a function of the deposition time. C) 25 dots with 10 s depositiontime each, the image size is 25 μm×25 μm, the colour map is shown inFIG. 1D. D) A linescan of the bottom row in image 2 C shows thereproducibility of the deposition and a fwhm of 1.0±0.1 μm.

[0022]FIG. 7: A) Fluorescent image of lines written in DNA by scanningthe sample under ion current distance control, 10 cycles with 35 μm/swere used for each line, the image size is 25 μm×25 μm. B) Squares of 20μm to 5 μm width were written one over each other to create a patternwith increasing intensities, the image size is 25 μm×25. C) The words“UNIVERSITY OF CAMBRIDGE” written on a 50 μm×50 μm area, the individualletters are 6-8 μm high.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0023] The present invention can be used to deposit any desired moleculeonto a wide variety of substrates. Examples of molecules that can bedeposited include biological molecules such as polynucleotides andpolypeptides (which terms are used to include oligonucleotides,oligopeptides and proteins). More particularly, the invention can beused to deposit long and short DNA, small molecules such as biotin,proteins such as streptavidin and antibodies, preferably proteins suchas enzymes and antibodies, DNA and other small molecules. Size is notcritical; most biological molecules are 5-10 nm, while long DNA is along thin cylinder. In certain cases, it may be desirable to coat thewall of the pipette at its tip, so that the molecules do not stick tothe glass.

[0024] The principle underlying the invention is illustrated in FIG. 1Aof the accompanying drawings. The pipette is immersed in solution abovethe surface onto which application will take place. Local applicationcan be controlled by the hydrostatic pressure applied and the size anddirection of the electrophoretic flow as well as the pipette-sampledistance. The molecules can be applied by deposition at a controlleddistance from the surface, or in a “tapping mode” where the pipetteamplitude of oscillation is increased so that the pipette comes veryclose or touches the surface for application. The surface is scannedunder the pipette for writing. The surface-pipette distance may becontrolled with nanometer precision using the modulated ion current.

[0025] The ionic strength in the micropipette may be different from thebath. Thus, there may be a clear change in conductance at the tip of thepipette on application, since the conductance will change from that ofthe bath solution to that of the micropipette solution. This allowsmonitoring of the application in situ. The applied voltage may bealtered to control application as well as the pipette-sample distance.The amount of molecules applied to the surface may be monitored bysensitive fluorescence spectroscopy, e.g. using an inverted microscope.Partial blocking of the ion current may be used to monitor delivery oflarge molecules.

[0026] The size of the features formed on the surface will depend, interalia, on the following parameters:

[0027] 1. The pipette diameter; this can be, for example, 10-100 nm.

[0028] 2. The pipette-sample distance; this may be controlled using themodulated ion current.

[0029] 3. The applied voltage; this controls the size and direction ofthe electrophoretic flow.

[0030] 4. The applied pressure (which leads to hydrostatic flow).

[0031] 5. The concentration of biomolecules in the pipette.

[0032] 6. The ionic strength; this can reduce the effective diameter ofthe pipette due to electrostatic interactions between charged moleculesand the surface.

[0033] 7. The duration and mode of the application, i.e. at fixeddistance or in tapping mode. In the tapping mode, it is possible thatthe number of applications can control the number of applied moleculeswithout increasing the feature size.

[0034] The molecules to be delivered are typically oligonucleotides(DNA) or oligopeptides. These molecules may be labelled. For example,the use of fluorescently labelled molecules allows direct visualisationof the features on the surface, e.g. using laser confocal microscopyduring or directly after application. A suitable microscope has twochannels for FRET or dual colour experiments. Gold-coated mica may allowhigher resolution AFM imaging.

[0035] Immobilisation of the delivered molecules on the surface to becoated can be achieved by a variety of means, physical and chemical.Examples are by biotin-(strept)avidin binding, or by charge.

[0036]FIG. 1A shows a two-barrel pipette, although one or more than 2barrels may also be used, depending on the molecule or molecules to bedelivered. For example, water-soluble molecules such as DNA or peptidescan be delivered from one barrel, and another molecule from the other,simultaneously or sequentially. Structures can be built up, by returningto the same point on the substrate surface and depositing differentmolecules, at the same position, and repeating this until the desiredstructure or molecule is provided at the appropriate location.

[0037] Controller software may be used to make lines or patterns ofmolecules on the surface of the substrate, so that work can be done onthe same surface but at different points. For instance, a series ofparallel lines of different DNA molecules can be written on the surfacefor cleavage. Both the control and experiment can then be performed, byapplying the appropriate enzyme from the pipette to a DNA line.

[0038] In order to load the solution or solutions for deposition intothe micropipette, a variety of methods may be used. In particularapplication of negative pressure or voltage of the appropriate polaritycan be used. In this fashion, with appropriate washing steps, thesolution in the pipette or one of the pipette barrels can be readilyinterchanged.

[0039] For the purpose of pipette delivery characterisation, the flow ofa fluorescently labelled DNA and protein such as streptavidin from thepipette can be determined as a function of pressure, applied voltage andpipette size. This is important for all subsequent experiments and alsouseful for functional mapping experiments on living cells. This will bedone into bulk solution using single molecule fluorescence, to directlyfollow the trajectories of fluorophor labelled molecules at the tip ofthe pipette. This has been done previously for 500 nm pipettes by Zanderet al, Chem. Phys. Lett. 1998, 286, 457. Surface modifications of thepipette to reduce non-specific adsorption can be adopted, e.g. usingsilane chemistry, if necessary.

[0040] A preferred embodiment of the invention is the production ofarrays. Whereas DNA arrays prepared by either spotting or delivery withpiezoelectric-driven micropipettes have a feature size that is typically200 μm in diameter or larger, the micropipette allows this size to bereduced to less than 100 nm. The arrays can be quickly built up bydelivering the DNA in a spotting action as the pipette is rapidly tappedover the glass surface. This may be performed under solution. The DNA istypically coupled to the surface using streptavidin-biotin interaction.Different combinations of DNA molecules may be delivered at one site orseparate sites using a double barrel pipette. This may be assayed usingtwo fluorescently labelled DNA molecules labelled with two differentfluorophors and imaged with a two colour confocal microscope.

[0041] The micropipette may also be used to set up protein arrays. Forexample, the surface may be coated with carboxy-terminated dextran(which has the advantage that it provides a softer landing for thepipette, thereby avoiding damage). The carboxyl groups can be locallyactivated by delivering the requisite coupling agents down anotherbarrel of the pipette. For example, in this way, NHS esters can be madeat a site immediately before the protein is delivered, or Ni²⁺ can bedelivered locally to facilitate NTA/His₆ interaction.

[0042] By means of the present invention, the feature size can be 1 μmor less. Further, protein delivery in solution and local application ofcoupling agent can bypass the problems described by Schreiber, supra. Itis worth noting that the smaller feature size requires smaller amountsof protein, which can be produced for example by in vitro translationsystems.

[0043] Having set up a protein array, a range of protein-protein, andprotein-small molecule, interactions can be explored. Generic(commercially available) systems to demonstrate the utility of thearrays are described by Schreiber, supra. For example, protein G(fluorescently labelled) and immunoglobin G are useful as a model forprotein-protein interactions, and fluorescently labelled biotin andimmobilised streptavidin as a model for protein-small moleculeinteractions.

[0044] It has previously been shown that DNA coated with silver can actas a relatively short nanowire (Braun et al, Nature 1998, 391,775). Anextended strand of dsDNA may be delivered to a surface by pushing it outof the pipette (like making a line of toothpaste on a surface). It hasbeen shown that the attachment of DNA to a negatively charged surface,such as glass, can be modulated by the presence or absence of divalentcations such as Zn²⁺. The DNA may be continuously or intermittentlypinned to the surface by delivering Zn²⁺ locally from another barrel ofthe pipette. If lengths of sticky-ended DNA are delivered sequentiallyin this way, extended strands of DNA may be assembled on a surface,which may initially be characterised by tapping mode AFM. Thesefragmented wires may subsequently be manipulated by ligases to stickthem together or endonucleases to create local breaks. The DNA may lieflat against a surface, but it is preferred that the DNA should act asan enzyme substrate which is able to locally dissociate between twopinned sites. Long lengths of fluorophor-labelled phage DNA may be usedfor these experiments, and the DNA may be visualised using intercalatordyes such as YOYO. The method of Braun et al, supra, may then be used toform silver wires by depositing silver on the surface-attached DNA.

[0045] Multi-component 3-dimensional structures made from proteinsor/and DNA can be assembled. The easiest way to exemplify this is usingthe biotin/strepatvidin system. For example, alternate layers ofstreptavidin and a biotinylated protein may be delivered onto a spot byalternately delivering them from different barrels of the pipette. Inthis way, a tower can be grown up from the surface. The streptavidin andprotein may be labelled with different fluorophors for visualisation.Likewise, it may be possible to graft together lengths of sticky-endedDNA orientated orthogonal to the surface, or alternate terminallybiotinylated DNA with streptavidin spacers.

[0046] International Patent Application No. PCT/GB02/01382 disclosesthat it is possible to deposit a single alpha toxin channel into acardiac myocyte and detect this using a patch clamp. The patch clamprecording is characteristic of a single alpha toxin channel. The presentinvention allows the development of a more general and flexible methodfor controlled delivery based on monitoring fluorescence or partialblocking of the current in the micropipette.

[0047] The following Examples illustrate the invention.

EXAMPLE 1

[0048] As suggested above, the invention makes it possible to delivertherapeutic DNA or proteins to a specific position on a living cell bymanipulating the pipette with precise distance control. Both shear forceand ion conductance are potential distance regulation methods. As afirst step to use the pipette for controlled delivery, it is essentialto characterize the flux of biomolecules in the nanopipette as theapplied voltage is altered. This Example reports the programmabledelivery of dye-labelled DNA molecules through a nanopipette byelectrical control. Quantitative measurements using single-moleculespectroscopy and fluorescence correlation spectroscopy (FCS) enable thedetermination of the number of molecules delivered.

[0049] Instrumentation

[0050] A home-made single-molecule confocal fluorescence microscope wasused; see Ying et al., J. Phys. Chem. B, 2000, 104, 5171-8; and Wallaceet al, PNAS USA 2001, 98, 5584-9. This microscope, equipped with apiezo-electric nanomanipulator, was used to view and position thepipette in the laser focus and also detect the fluorescence signal fromdye-labelled DNA. An argon ion laser (Ion Laser Technology 5490AWC-00)at 488 nm was used as the excitation source. The collimatedlinear-polarized laser beam was directed through a dichroic mirror tothe inverted microscope (Nikon Diaphot 200) and focused by an oilimmersion objective (Nikon Fluor 100×, NA 1.30). Fluorescence wascollected by the same objective and imaged onto a 100 mm pinhole toreject out of focus fluorescence and other background. The fluorescencewas then filtered by a bandpass and a longpass filter (Omega Optical535AF45 and OG515) before being focused onto an avalanche photodiode,APD (EG&G SPCM AQR-141). Output from the APD was coupled to a PCimplemented multichannel scalar card (EG&G MCS-Plus). A bent nanopipettewas mounted to a home-made nanomanipulation system consisting of amodular focusing unit (Nikon 883320), a mechanical translation stage anda three-axis piezoelectric translation stage (Piezosystem JenaTritor38). The modular focusing unit and the mechanical translationstage were used for coarse positioning of the nanopipette. Thepiezoelectric stage was used for fine adjustment.

[0051] Glass pipettes with inner radii around 50 nm were routinelyfabricated using a laser-based pipette puller (Sutter InstrumentP-2000). A voltage was applied to the nanopipette through two Ag/AgClelectrodes, one in the bath and the other inside the pipette, serving asthe working and reference electrodes respectively. The ion current flowthrough the pipette was amplified by a high impedance amplifier andmonitored by an oscilloscope.

[0052] Materials and Experimental Procedure

[0053] A DNA oligonucleotide (SEQ ID NO. 1) was synthesized by Cruachem(Glasgow, UK) and was purified twice by HPLC. The 3′ end was modified byRhodamine Green (Molecular Probes). The concentration of thedye-labelled DNA was determined by UV-Vis absorption at 260 nm, and theabsorbance at 504 nm was used as an internal reference. The purity ofthe sample is better than 95% based on this internal check.

[0054] 100 nM DNA solution was back-filled to the bent nanopipette by amicrofiller (World Precision Instruments Microfil 34). Blockage of thenanopipette was avoided by filtering the solutions through a 20 nmfilter. A coverglass bottomed dish (Willco Wells GWST-1000) containing2-3 ml solution was used as the bath. The pipette tip was placed 5 to 10μm above the dish surface. Identical buffers (10 mM Tris-HCl, 100 mMNaCl) were used both in the pipette and in the bath. For single-moleculedetection in the nanopipette, 5 nM DNA solution was used and the laserpower was 0.25 mW. Fluorescence correlation measurements were performedby correlating the MCS signal in real-time using our own softwaredeveloped in Labview environment (National Instruments EvaluationVersion 5.0).

[0055]FIG. 1B is a schematic view of the experimental setup. A videoimage of the nanopipette and fluorescence spot near the tip, excited bya focused 488 nm laser beam, showed a spot size of around 500 nm, biggerthan the inner diameter of the pipette due to the limitation offar-field optical resolution, which is consistent with the probe volumemeasurement using FCS. When the laser focus was moved into the pipette,an elliptical fluorescence spot with length around 1.5 mm was observed.

[0056] When a voltage was applied across the two electrodes, thepotential drop occurred almost entirely in the tip region, due to theconical geometry of the pipette. The applied electric field was highlynon-uniform and was located very close to the tip (the field strengthdrops to less than 1% of the maximum at 10 μm distance away from thetip). High field strength (several kV/cm) was easily achieved near thetip with very low applied potential (hundreds of mV), in contrast withthe standard capillary electrophoresis method which requires a highvoltage source.

[0057] When the applied voltage was modulated with a square wave at acontrolled frequency using a function generator, highly repetitivepulsed delivery of DNA was achieved, as shown in FIG. 2. Optimal pulseswith high on/off ratio could be obtained by manipulating the modulationamplitude and the duty cycle.

[0058] The number of molecules delivered was estimated based onsingle-molecule photon burst size and characteristic diffusion timemeasurements and will be discussed below. In FIG. 2A, about 150±20molecules were detected per pulse when modulating at 10 Hz from −0.1 Vto 0.6V with positive voltage duration of 30 ms; while in FIG. 2B, about460±50 molecules were detected per pulse when modulating from −0.1V to1.5V with positive voltage duration of only 5 ms. In the latter case,the result suggests non-linear focusing of the DNA molecules towards thenanopipette tip because the temporally averaged mean field is negative,which would drive the macroions deep into the pipette if the systemresponse were linear, instead of exiting the pipette as was observed.The mechanism may be similar to non-linear electrophoresis.

[0059] To estimate the number of molecules delivered per pulse, singlemolecule measurements were carried out near the nanopipette tip. Inthese experiments, the laser focus was centered at the opening of thenanopipette. As the diffraction-limited spot size was much bigger thanthe pipette radius, most of the molecules coming out of the pipette tipcould be detected. Single-molecule burst size analysis gave the meannumber of photons detected from each molecule as 35 per millisecond atthe excitation laser power of 250 μW. Single-molecule measurements atdifferent applied voltage provided a clear view of how the burstfrequency changes with voltages; see FIG. 3A. Pulsed delivery of DNAmolecules with single-molecule detection was demonstrated in FIG. 3B.About 20 individual molecules were detected per cycle.

[0060] The S/N ratio was high (background 0.7 counts/ms, maximumsingle-molecule burst 150 counts/ms). There was no seriousback-scattering of the tightly focused laser beam on the glass wall eventhough there is significant difference in the index between water andglass media.

[0061] Fluorescence correlation measurements were carried out inside thetip of the pipette and in the open volume, to determine the diffusionconstant of DNA and to address how the glass surface impedes thediffusion of ss-DNA molecules. The diffusion coefficient of thedye-labelled ss-DNA in free solution is 1.7×10⁻⁶ cm²sec⁻¹, compared tothat of 8.1×10⁻⁸ cm²sec⁻¹ with a temporal exponent of 0.81 in thenanopipette. The anomalous diffusion of DNA is thought to be a result ofthe confinement of the glass wall. A factor of about 20 longer diffusiontime was observed, for confined short ss-DNA molecules. When a potentialof several hundred millivolts was applied, there was no significantdifference in the autocorrelation curve, indicating that the flow rateis quite slow compared with the diffusion. However, a large decrease (˜a factor of 20) in the correlation amplitude was observed when thevoltage changed from 50 mV to 300 mV. This suggests a substantialconcentration enhancement in the tip region because the correlationamplitude is inversely proportional to the number of molecules in theconfocal volume. Given the mean diffusion time through the laser focusand the mean count rate of single-molecule fluorescence, it is estimatedthat on average 28 counts can be detected with laser excitation power100 μW for an individual rhodamine green-labelled DNA molecule passingthrough the laser focus, when the beam is centered on the opening of thepipette. In this calculation, the possible fluorescence lifetime hasbeen neglected, as has the spectrum change due to the effect ofconfining microcavity formed in the nanopipette tip.

[0062]FIG. 4A shows the pulsatile delivery patterns generated atdifferent modulation voltages. A small negative potential is required toachieve high modulation depth in the fluorescence signal. At lowvoltages, a slow rise and a fast decay of the fluorescence are observed.At high voltages, the decay becomes much slower when the voltage isreversed, indicating that DNA molecules continue to exit the pipettedriven by the local concentration gradient. The concentration of DNA inthe tip region could be increased by two orders of magnitude by applyinga suitable sign voltage pulse. The duration of the pulse could be usedfor controlled delivery of DNA through the nanopipette. As a result, itwas possible to observe highly controlled delivery of DNA through thenanopipette. For example, a peak fluorescence intensity of 200 countsper 0.2 ms corresponds to 72 molecules in the detection volume. Assumingthe effective confocal volume on the order of 0.01 fL for a 50 nmpipette, then in the focused region, the concentration would be around12 μM. However, the concentration distribution in narrow conicalstructure was expected to be heterogeneous when the effect of surfacecharge is considered. The delivery efficiency per modulation cycle as afunction of voltage is shown in FIG. 4B. A linear relationship was foundwhen the voltage is larger than 0.5V. The linear dynamic range for thenumber of molecules detected is between 100 and 2000 in pulsed deliveryusing 100 nM sample. Delivery from a few molecules to a few thousandmolecules per pulse can be easily controlled by changing the modulationamplitude and sample concentration. In continuous delivery mode, alinear relationship was also observed at applied potential between 0.2Vand 1.0V. Even higher potential induces a significant trapping effect,and the system response is non-linear.

[0063] The frequency response of the nanopipette system for pulsatiledelivery was also examined. At frequencies higher than 200 Hz, it wasdifficult to observe the change in count rate as the number of countsbecomes very small in a short collection time. As such, the fluorescenceautocorrelation function was used to reveal the fluctuation in timedomain. At 1000 Hz, deep modulation can still be achieved. At 5000 Hz,little modulation was observed. Therefore, pulsatile delivery may beoperated up to 1000 Hz.

[0064] In summary, Example 1 shows that pulsatile delivery of DNAmolecules can be achieved in a simple and reliable way using ananopipette. This ultra-sensitive technique is able to deliver a fewmolecules per pulse. The combination of surface modification of theinner wall and reductions in the size of the pipette, may allow moreprecise control of the delivery of not only the nucleic acid but proteinand other nanoparticles as well. The technique may open new routes foraccurate antisense drug delivery into a single living cell. Furthermore,this technique can easily be combined with SICM technology and may bedeveloped as a versatile nanopen for micro and nanofabrication ofbiochips in aqueous environments.

EXAMPLE 2

[0065] In this Example, the pipette is used for the delivery ofbiotinylated and fluorophore-labelled DNA to a streptavidin-coatedsurface.

[0066] First, the flow of single-stranded DNA (see above) out of thenanopipette was studied, using fluorescence measurements at the tip ofthe pipette; see FIG. 5A. The same rhodamine green-labelledoligonucleotide as in Example 1 was used.

[0067] Nanopipettes were routinely made by a laser-based pipette puller(Sutter Instrument P-2000). Fluorescence was measured using a home-madesingle-molecule confocal fluorescence microscope, based on a NikonDiaphot 200 with an oil immersion objective (100×, NA 1.30) and anavalanche photo-diode detector (EG&G SPCM AQR-141). 100 W of the 488 nmradiation from an argon ion laser was used for excitation.

[0068] Single molecule counting was used to estimate the number of DNAmolecules leaving the pipette, as described above. It was found that theflux of DNA could be controlled by a voltage applied between thecounter-electrode and an electrode inside the pipette. When thecounter-electrode was at a negative potential relative to the pipette,negligible DNA flowed out of the tip. On application of a positivepotential, DNA flow occurred and the flux was linear with appliedpotential; see FIG. 6B. This demonstrated that there is fine control ofthe rate of DNA delivery by controlling the applied potential over aconvenient and modest voltage range.

[0069] Next the DNA was deposited on a streptavidin-coated glasssurface, using ion conductance control of the pipette sample distance;see FIG. 5C. More particularly, glass-bottomed dishes (WillCo, WellsB.V., NL) were coated with streptavidin by BioTeZ Berlin-Buch GmbH. Thebiotin-binding capacity of these coatings is 280 fmol/mm² (68 pg/mm²).Writing experiments were performed using an inverted microscope (EclipseTE 200, Nikon) with two piezoelectric xyz-stages, one to move thepipette and one to scan the sample. The tip was modulated ±50 nm and themodulated ion current, recorded by a lockin amplifier, was used fordistance control. Phosphate buffer solution (10 mM Na₂HPO₄, 137 mM NaCl)was used in the bath and in the pipette containing 100 nM ssDNA.

[0070] The sample piezo stage carrying the glass slide was manipulatedwith nanometer precision by manually changing the input voltages. Inthese experiments, the voltage applied to the counter electrode was keptconstant at 600 mV, so that the flux out of the pipette was about 4000molecules/s. FIG. 6A shows spots of DNA where the pipette was left closeto the surface for increasing lengths of time, from 5 s to 160 s.Streptavidin-biotin binding immobilizes the DNA at the surface andfluorescence of the rhodamine-labelled DNA was detected by scanningconfocal microscopy. The detection was done on the same instrument with0.5 μm optical resolution (FIG. 5D). Images were recorded under 30 μWillumination at 488 nm. The fluorescence signal was detected using anoil-immersion objective (100×, NA=1.25) and a photomultiplier tubedetector. A dicroic beamsplitter, longpass filter and a 50 μm pinholewere used to separate the fluorescence signal and to reject backgroundsignal and laser scattering.

[0071] Spots of the deposited DNA are clearly seen in the image. Theoverall intensity increases linearly with the deposition time (FIG. 6B),whereas the peak intensity shows a saturation effect with a halftime of40 s. As a result, the detected full width of half maximum (fwhm) of thebell-shaped intensity profile increases from 1.1 μm to 3.0 μm. FIG. 6Cshows an array of 25 spots, deposited for 10 s each. The measured fwhmis 1.0±0.1 μm and the intensity varies only ±6% which shows the goodreproducibility of the method.

[0072] By scanning the stage with computer control, a more complexpattern can be produced. FIG. 7A shows lines separated by 3 μm, againthe width is 1.0 μm. In FIG. 7B, squares were written one over another,so that areas of different intensity were produced, demonstrating thepossibility of not only the formation of patterns but also of writing in“grey-scale”. Moving the pipette by manually changing the input voltagesof its piezo stage allows the writing of letters in DNA of only 6-8 μmsize onto the streptavidin surface; see FIG. 7C.

[0073] Since the operation is in ionic solution, the feature size willdepend on the distance the pipette is held from the surface anddiffusion. The feature size may be extracted using the steady stateconcentration profile derived for ultra-microelectrodes in the diffusionlimited case, as described by Bond et al, J. Electroanal. Chem. 1988,245, 74. For a 100 nm pipette held 75 nm from the surface, the fwhm ofthe features would be expected to be about 300 nm. The observed featuresize is a factor of 3 larger and this may be due to 2D diffusion of theDNA on the surface and the low density of streptavidin sites on theglass surface. However, the feature size is at least one order ofmagnitude smaller than that obtained for DNA deposition in air, andcontrol of the amount of DNA delivered is simple.

[0074] Sub-micron features can be achieved by optimisation of themethod, including the use of finer pipettes, using higher densitysurfaces and covalent attachment chemistry. However, the current featuresize is well-suited for optical read-out. This method is also simple andoperates under physiological conditions. It is straightforwardlyextendable to other biological molecules, as indicated by thepreliminary experiments on protein G and streptavidin, and is applicableto enzymes and antibodies. It may also be used to produce complexbiological structures on surfaces.

EXAMPLE 3

[0075] A protein array was written using the same experimental setup asfor the DNA array. A solution of 100 nM fluorescently-labelled protein Gin phosphate buffer solution (10 mM Na₂HPO₄, 137 mM NaCl) was backfilledinto the pipette and the same phosphate buffer was used in the bath.Using a pipette of 100 MΩ resistance, the protein exits out of thepipette on application of a voltage of −500 mV to the Ag/AgCl electrodein the bath. The surface of a microscope coverglass was treated withaminosilane to provide a positive surface charge. The pipette wascontrolled over this surface at a distance of about 75 nm using themodulated ion current as a feedback signal. Pipette modulation was about±50 nm. Moving the pipette under these conditions over the surfaceallows the protein to be deposited onto the glass surface. The amount ofprotein delivered to the surface depends on the time the pipette is heldat one position. It was detected with 10 μW excitation at 488 nm and thesame confocal fluorescent microscope as used for the DNA array.

EXAMPLE 4

[0076] This Example illustrates modifying a glass nano-pipette to reduceprotein adsorption.

[0077] 3-[Methoxy(polyethyleneoxy)propyltrimethoxysilane](PEG-Silane, >90%, av. FW 460) was purchased from ABCR (Germany), andanhydrous 1,4-dioxane was purchased from Aldrich.

[0078] (1) Freshly pulled glass nano-pipettes were dipped into a freshlyprepared 5% PEG-silane (v/v in anhydrous 1,4-dioxane) solution for 5-10mins. The pipettes were then washed 20 times in pure 1,4-dioxane bydipping the tips into and then pulling out from the pure 1,4-dioxanesolution. The dipping and pulling cycle results in 1,4-dioxane enteringinto and coming out of the tip region of the pipette as observed via aconfocal microscope.

[0079] (2) The modified tips were then baked in an oven at 100° C. atambient atmosphere for 2 hrs. The baking process is necessary to attachthe PEG-silane covalently to the glass surface of the tip.

[0080] (3) After the baking, the pipettes were used to deliver Alexa 488modified streptavidin (Molecular Probes) onto the biotin-modified glasssurface at a concentration of 100 or 10 nM in PBS buffer (10 mMphosphate, 150 mM NaCl, 2 mM NaN₃, pH 7.20) to form sub-micrometer sizedprotein arrays.

[0081] As the surface property of the pipettes is difficult tocharacterize, a control experiment on the water wettability of glasscover slips was carried out. The contact angle for pure water before themodification was ≦5°. After the modification, the contact angleincreased to ca. 37±1°, in good agreement with the literature value ofPEG-terminated surfaces, suggesting the successful modification of theglass surface.

1. A method for the production of an array of molecules immobilised on asubstrate, which comprises the deposition of the molecules from amicropipette containing them, onto the substrate, in a liquidenvironment, wherein the distance of the micropipette from the substrateis controlled in response to the ion current in the liquid.
 2. Themethod according to claim 1, wherein the molecules are polynucleotides.3. The method according to claim 1, wherein the molecules arepolypeptides.
 4. The method according to claim 1, wherein themicropipette is oscillated substantially normal to the surface of thesubstrate, and the said distance is controlled by modulation of thesurface of the ion current.
 5. The method according to claim 1, whereinthe micropipette is oscillated substantially normal to the surface ofthe substrate, with large or increased amplitude, so that themicropipette is at or very close to the surface of the substrate, ondeposition.
 6. The method according to claim 1, wherein the micropipettehas two or more barrels.
 7. The method according to claim 6, whereindifferent molecules are delivered from each barrel.
 8. The methodaccording to claim 1, which comprises delivering different molecules tothe same point on the substrate, to build up a structure or molecule ata desired location.
 9. The method according to claim 1, wherein thedeposition is controlled by the application of pressure or voltage. 10.The method according to claim 1, wherein the feature size of the arrayis less then 1 μm.
 11. The method according to claim 10, wherein thefeature size is 10 to 100 nm.
 12. An array of biological moleculesdeposited on a substrate, wherein the feature size is less than 1 μm.13. The array according to claim 12, wherein the feature size is 10 to100 nm.
 14. The array according to claim 12, obtainable by a methodaccording to claim
 1. 15. The array according to claim 13, obtainable bya method according to claim
 1. 16. The method according to claim 1,wherein the deposition is controlled by the application of pressure andvoltage.